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PubMed2002

Hybrid tetanus toxin C fragment-diphtheria toxin translocation domain allows specific gene transfer into PC12 cells.

To study the mechanism by which genes can efficiently be transferred into specific cell types, we have constructed several novel, single-chain multicomponent proteins by recombining the nontoxic C fragment of tetanus toxin and the translocation domain of diphtheria toxin together with the DNA-binding fragment of GAL4 transcription factor, for transportation of plasmid DNA into neuronal cells. The C fragment of tetanus toxin provided neuronal selectivity, the translocation domain of diphtheria toxin permitted endosomal escape, and the GAL4 domain provided binding to DNA. To assess the cellular tasks of each component in gene transfer, different combinations of these fragments were produced by polymerase chain reaction, expressed in Escherichia coli, and purified under native conditions from the soluble proteins. We show that only fusion proteins bearing the C fragment of tetanus toxin bind to gangliosides and, followed by their specific binding to differentiated PC12 cells, are internalized within 10 min. These proteins delivered the green fluorescence protein gene to PC12 cells, with the highest transfection efficiency achieved with proteins containing both the C fragment and the translocation domain. Addition of chloroquine elevated the transfection efficiency, which was further increased by incorporation of a nuclear localization signal in the delivery system. In addition, the effect of different DNA-condensing materials (poly-L-lysine, protamine, lysine(n=8)-trytophan(n=2)-lysine(n=8)) on gene transfer was investigated.

Amino Acid Sequence↗
PubMed1980

A hybrid toxin containing fragment A from diphtheria toxin linked to the B protomer of cholera toxin.

We have constructed and characterized a hybrid toxin containing the A chain of diphtheria toxin linked via a disulfide bridge to the B protomer of cholera toxin. Cholera toxin B protomer, previously derivatized with 4-5 cystaminyl groups per pentameric protomer, was reacted with reduced diphtheria toxin chain A to give the desired hybrid, containing an average of 2 molecules of diphtheria toxin chain A per cholera toxin B protomer. A concentration of 0.3 nM hybrid inhibited protein synthesis by 50% in 24 h in several cultured cell lines; thus the hybrid was about 10-fold more toxic than of a (diphtheria toxin chain A)-SS-(concanavalin A) conjugate described previously. Evidence was obtained that toxicity of the hybrid was dependent on the functional contributions of both the diphtheria toxin chain A and cholera toxin B protomer moieties.

Animals↗
PubMed1981

Methylamine stimulates the action of ricin toxin but inhibits that of diphtheria toxin.

The action of ricin toxin was stimulated by addition of methylamine or some other amines, as shown by measuring the inhibition of protein synthesis of cultured cells by the toxin. Under the same conditions, however, the action of diphtheria toxin was completely inhibited by the amines. In a cell-free protein-synthesizing system, methylamine had no effect on the action of the A chain of ricin toxin and fragment A of diphtheria toxin. Studies on the interactions of 125I-labeled toxins with cells revealed that methylamine did not alter toxin-receptor bindings, but affected the entry of the toxins into the cells. Studies were also made on the effects of methylamine on the actions of two hybrid toxins, formed from a subunit of Wistaria floribunda lectin and fragment A of diphtheria toxin and the A chain of ricin toxin, respectively. Results suggested that the processes of entry of ricin toxin and diphtheria toxin, or at least parts of these processes, are different.

Cell-Free System↗
PubMed1986

Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin.

When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin.

Adenosine Triphosphate↗
PubMed1999

In vitro-translated diphtheria toxin A chain inhibits translation in wheat germ extracts: analysis of biologically active, caspase-3-resistant diphtheria toxin mutants.

The diphtheria toxin A chain (DTA) is a potent cytocidal agent that inactivates elongation factor 2. This activity of DTA inhibits protein synthesis and rapidly leads to cell death through apoptosis. In this paper, we have developed a simple in vitro assay for DTA activity in which in vitro-translated DTA is used to inhibit the translation of proteins in wheat germ extracts. Inhibition of translation by DTA is dependent on cofactor NAD+, and the analysis of an attenuated DTA mutant indicates that this in vitro assay accurately reflects the in vivo activity of DTA. We have also identified aspartic acid at residue 8 (Asp-8) of DTA as a site of cleavage by the cell-death protease caspase-3. Cleavage of DTA by caspase-3 inactivates its ability to inhibit translation in wheat germ extracts. Conservative mutations at Asp-8 render DTA resistant to cleavage by caspase-3, but only slightly affect the ability of DTA to inhibit translation in vitro. Moreover, caspase-3-resistant DTA mutants are toxic in cells in tissue culture. The in vitro assay that we describe here will be useful for the rapid analysis of DTA activity and the development of DTA mutants with altered biological properties that may be of therapeutic value. Lastly, these studies serve as a prototype for the creation of caspase-resistant effector molecules.

Amino Acid Sequence↗
PubMed2001

Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.

BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.

Animals↗
PubMed1993

The role of proline 345 in diphtheria toxin translocation.

Diphtheria toxin (DT) can translocate across endosomal membranes in response to low pH. Buried hydrophobic domains localized in the 37-kDa toxin B chain become exposed in response to acidic conditions and are thought to participate in the membrane translocation process. The crystal structure of DT has revealed a structurally distinct translocation domain composed of nine alpha-helices with their interconnecting loops (Choe, S., Bennett, M., Fujii, G., Curmi, P., Kantardjieff, K., Collier, R., and Eisenberg, D. (1992) Nature 357, 216-222). Two of these alpha-helices, TH8 and TH9, are unusually apolar and constitute the central core of the translocation domain. It has been proposed that these domains and the highly charged interconnecting loop undergo a conformation change under acidic conditions producing a dagger-like structure capable of inserting into the membrane thus initiating the translocation process. Proline 345 occupies a strategic location at the end of the TH8 alpha-helix. Proline residues have the ability to undergo a cis-trans isomerization reaction and because of this have been proposed to play a role in the conformational change that is a prerequisite for toxin translocation. The role of the proline at position 345 in membrane translocation was investigated. Pro was mutagenized to Glu and to Gly using a two-step recombinant polymerase chain reaction procedure, and the mutant proteins were expressed in vitro. Glu, an alpha-helix former, and Gly, an alpha-helix breaker, were selected for mutagenesis to distinguish between a structural role for Pro as an alpha-helix breaker and alternative roles, perhaps involving cis-trans isomerization-related conformational changes. Replacing Pro at position 345 with Glu or Gly resulted in a 99% reduction in toxicity to Vero cells. The enzymatic and binding activity of the toxin were not altered by the mutations. Instead, the reduction in toxicity is due to decreased translocation ability, suggesting that the Pro at position 345 plays a specific role in toxin membrane translocation.

Amino Acid Sequence↗
PubMed2006

Protein translocation by bacterial toxin channels: a comparison of diphtheria toxin and colicin Ia.

Regions of both colicin Ia and diphtheria toxin N-terminal to the channel-forming domains can be translocated across planar phospholipid bilayer membranes. In this article we show that the translocation pathway of diphtheria toxin allows much larger molecules to be translocated than does the translocation pathway of colicin Ia. In particular, the folded A chain of diphtheria toxin is readily translocated by that toxin but is not translocated by colicin Ia. This difference cannot be attributed to specific recognition of the A chain by diphtheria toxin's translocation pathway because the translocation pathway also accommodates folded myoglobin.

Colicins↗
PubMed1989

A point mutation of proline 308 in diphtheria toxin B chain inhibits membrane translocation of toxin conjugates.

Diphtheria toxin (DT) is a soluble protein that translocates across hydrophobic lipid bilayers in response to low pH. The translocation activity of DT has been localized to the 40-kDa toxin B chain and can be expressed independently of the C-terminal receptor binding site. Buried hydrophobic domains in DT are thought to participate in the membrane translocation process. We have identified a mutant form of DT, CRM 102, that has a point mutation at position 308 (Pro----Ser) within one of these hydrophobic domains. CRM 102 conjugated to a monoclonal antibody against the T cell receptor, the transferrin receptor, or transferrin itself is approximately 10-fold less toxic than native DT or a control DT mutant, CRM 103, linked to the same binding moieties. Direct measurement of membrane translocation activity by exposure of cells to low extracellular pH demonstrates that CRM 102 conjugates express only 10% of the translocation activity of the control toxin conjugates. However, when CRM 102 or 102 conjugates bind and kill cells via the DT receptor, no reduction in membrane translocation activity is observed. The defect in CRM 102 is not evident in the presence of 20 mM NH4Cl. The defect in translocation also has no effect on the ratio of the lag time before protein synthesis inhibition begins to the rate of protein synthesis inhibition. Thus, the proline-serine substitution at position 308 disrupts the membrane translocation process and distinguishes between two routes of DT entry: DT receptor-mediated entry and entry mediated by alternate receptors.

Animals↗
PubMed1998

Diphtheria toxin translocation across endosome membranes. A novel cell permeabilization assay reveals new diphtheria toxin fragments in endocytic vesicles.

By using cells overexpressing diphtheria toxin (DT) receptor and a novel method of permeabilizing the plasma membrane with a bacterial pore-forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles. The translocation competence of DT proteins with mutations in the transmembrane domain is consistent with their cytotoxicities. The charge-reversal mutants E349K and D352K do not translocate their fragment A to the cytosol, whereas D352N is partially competent. ADP-ribosyltransferase activity of fragment A is not required for translocation. Novel fragments of DT with apparent molecular masses of 28 and 35 kDa were detected in endocytic vesicles. The 28-kDa fragment consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment contains part of fragment B and may be complementary to the 28-kDa fragment. Time course studies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We present a model for translocation of fragment A that incorporates the observations made using the novel permeabilization method.

Animals↗
PubMed1994

Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which up-regulates functional receptors and diphtheria toxin sensitivity.

DRAP27, the monkey homolog of human CD9 antigen (DRAP27/CD9) and diphtheria toxin receptor (DTR) were expressed in mouse L cells. L cells transfected transiently with both DRAP27/CD9 and DTR cDNA bound approximately 10 times more diphtheria toxin (DT) than cells transfected with DTR alone. Stable L cell transfectants expressing both DTR and DRAP27/CD9 (LCH-1 cells) had 15 times more cell surface DT-binding sites and were 20 times more sensitive to DT than were stable L cell transfectants expressing DTR alone (LH-1 cells). Increased DT-binding and DT sensitivity were not due to increased DTR transcription or increased cell surface DTR protein. Co-immunoprecipitation of DRAP27/CD9 with DTR and chemical cross-linking suggest a tight association of these membrane-bound proteins. In addition, the identity of DTR and a growth factor (HB-EGF) was established. Immobilized DT specifically adsorbed HB-EGF precursor solubilized from transfected L cells and [125I]DT bound to immobilized recombinant HB-EGF. We conclude that DRAP27/CD9 associates tightly with DTR/HB-EGF and up-regulates the number of functional DTRs and DT sensitivity, and that HB-EGF is identical to DTR.

Amino Acid Sequence↗
PubMed1992

Construction of a diphtheria toxin A fragment-C180 peptide fusion protein which elicits a neutralizing antibody response against diphtheria toxin and pertussis toxin.

A genetically engineered gene fusion was constructed which encoded a nontoxic derivative of the A fragment of diphtheria toxin joined to the C180 peptide of the S1 subunit of pertussis toxin. The product of this gene fusion, termed the DTA-C180 protein, was purified from the periplasm of Escherichia coli to approximately 80% purity. The DTA-C180 protein possessed an apparent molecular weight of 43,000 by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DTA-C180 protein was cleaved into two tryptic peptides, which migrated with apparent molecular weights of approximately 22,000. One tryptic peptide reacted with diphtheria antitoxin, while the other tryptic peptide reacted with anti-C180 peptide immunoglobulin G. The DTA-C180 protein did not inhibit protein synthesis or stimulate clustering morphology in Chinese hamster ovary cells. The DTA-C180 protein elicited an immune response, in guinea pigs, against both the DTA and C180 peptide components of the fusion protein, with alum being a more efficient adjuvant than Freund's adjuvant for eliciting neutralization titers. Neutralization titers elicited by DTA-C180 protein were weaker than those elicited by diphtheria toxoid and pertussis toxin 9K/129G, a genetically engineered double mutant of pertussis toxin. Three doses of DTA-C180 protein yielded a neutralization titer of 1/750 against pertussis toxin in Chinese hamster ovary cells and a neutralization titer of 1/50 against diphtheria toxin in Vero cells. This is the first report of a protein derived from a recombinant S1 subunit that elicits a neutralizing titer against pertussis toxin.

Adenosine Diphosphate Ribose↗
PubMed1992

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

Amino Acid Sequence↗
PubMed1985

Diphtheria toxin promoter function in Corynebacterium diphtheriae and Escherichia coli.

The expression of the diphtheria tox228 gene encoding the nontoxic, serologically related CRM228 mutant diphtheria toxin has been analyzed in Corynebacterium diphtheriae and Escherichia coli. The diphtheria toxin promoter has been used to direct the expression of beta-galactosidase in E.coli, and the efficiency of promotion has been compared to that obtained with the lac promoter. Expression in C.diphtheriae is known to be dependent on the absence of iron, and we present for the first time direct evidence that this regulation occurs at the level of transcription. The 5' end of toxin mRNA maps at the same position in C.diphtheriae and E.coli, suggesting identical sequences to be recognized by C.diphtheriae and E.coli RNA polymerase. The diphtheria toxin promoter carries at position -34 a TTGATT sequence closely related to the E.coli -35 consensus sequence and in the -14 to -8 region a set of overlapping sequences with complete or partial homology to the E.coli -10 consensus sequence.

Base Sequence↗
PubMed2003

Metal stoichiometry and functional studies of the diphtheria toxin repressor.

Diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor in Corynebacterium diphtheriae. DtxR is an iron sensor; metal-bound DtxR represses transcription of genes downstream of the tox operator. Wild-type DtxR [DtxR(wt)] and several mutant forms were overexpressed and purified from Escherichia coli. DtxR was isolated without bound metal. Metal reconstitution gave a binding stoichiometry of 2 per monomer for DtxR(wt) and 1 per monomer for DtxR(H79A) and DtxR(M10A). DNA binding of DtxR(H79A) and DtxR(M10A) indicates that metal site 2 is essential for activity. Metal binding lowers the dimerization K(d) of DtxR from low micromolar to 33 nM. Gel electrophoretic mobility-shift assays show that Fe(2+) and not Fe(3+) activates DtxR for DNA binding. This finding suggests that gene regulation by DtxR may be sensitive not only to iron levels but also to redox state of the iron. Mutations in the tox operator sequence indicate that DtxR dimers binding to DNA may be highly cooperative.

Bacterial Proteins↗